Project 3 focuses on the development of metabolomic signatures of past radiation exposure in biofluids such as urine and serum. These biomarkers are particularly useful because of the potential for non invasive sample acquisition and the long metabolomic signal lifetime - days or weeks after exposure. Metabolomic signatures developed to date have been predictive of both dose and late health outcome. The themes here are ?Beyond Simple Exposures?, ?Beyond Dose?, ?Beyond Model Systems? and ?Optimized Biomarker Integration?, which are motivated by the variety of different exposure scenarios and countermeasure needs. Beyond Simple Exposures: While most radiation biodosimetry studies have involved photons at intermediate dose rates, realistic exposure scenarios to which individuals will be exposed after an IND may include: mixed neutron+photon exposure, very high dose rates, variable low dose rates, and partial body exposure. This CMCR uses unique irradiation facilities designed to simulate these exposures, and these will be used to assess if a reference metabolomic signature can reconstruct the dose - or if additional metabolites are required to identify different exposure scenarios. Beyond Dose: Metabolomic biomarkers have been useful not only for reconstructing past radiation dose but also for predicting photon-induced pulmonary death. Here these predictive capabilities will be assessed following mixed neutron+photon exposures. Mechanistically, a focus will be on radiation-induced senescent cell signaling, which is a likely player in the development of late pulmonary injury. The contribution of radiation- induced senescent cell signaling will be evaluated in photon vs. mixed neutron+photon induced late lung injuries, and how it affects the predictive signature of these injuries. Beyond Model Systems: In that most biodosimetry studies are of necessity conducted in animal models, this theme addresses the link between metabolomic biomarkers of radiation exposure in animals vs. humans. Although many relevant exposures cannot be directly investigated in humans, samples from photon-exposed TBI patients can help guide the translation of biodosimetry assays from animal models. A very large metabolomic database from TBI patients, mice, and NHPs will be analyzed to test the hypothesis that there is a common radiation-responsive metabolomics signature across all three species. Optimized Biomarker Integration: The three different biomarker systems in this CMCR program cytogenetics, gene expression, and metabolomics reflect different balances of capabilities in terms of throughput, time-to-result, dose reconstruction, exposure scenario identification and radiosensitivity prediction. Our common goal is to identify their optimal integrated usage in a wide variety of different large-scale exposure scenarios. As results emerge from this Project, they will be used as input to optimize decision trees to determine which assay, or combination of assays, will be most effective in each radiation event scenario.